Many transporters including neurotransmitter transporters use the movement of coupled ions and charge to drive the accumulation of substrate against a concentration gradient. Conversely, the movement of coupled ions and charge can be used to identify the substrates for orphan transporters. We have recently used pH imaging and electrophysiology to show that several orphan transporters exhibit the properties of classical amino acid transport systems N and A. The functional characteristics and location of these proteins further suggest a role in the glutamine-glutamate cycle that replenishes glutamate (and GABA) released from nerve terminals during synaptic transmission. The coupling of system N to H+exchange and the electrogenic nature of system A have also suggested that pH imaging and electrophysiology might be used to study intracellular transporters, which often couple to H+. Indeed, we have recently observed pH changes produced by substrates in cells expressing a lysosomal transporter mislocalized to the plasma membrane. The long-term objective of this proposal is to understand the function of intracellular transport proteins implicated in synaptic transmission and development. The strategy is to mislocalize these proteins on the cell surface so that we can use pH imaging and electrophysiology to determine their function. In terms of specific aims, we propose to 1) Knock out glutamine synthetase in the nervous system. Although the glutamine-glutamate cycle was proposed decades ago, its role in transmitter release remains unclear. We will therefore use cre recombinase to knock out the gene for glutamine synthetase specifically in the nervous system, and determine the effect on synaptic transmission. 2) Identify the substrates for synaptic vesicle protein SV2A. Among the first synaptic vesicle antigens to be identified, SV2 proteins strongly resemble a large family of transporters. Knock-out mice for SV2A alone show a severe phenotype. However, the function of SV2 remains unknown. We will now use biophysical approaches to characterize the function of SV2 mislocalized at the plasma membrane. 3) Determine the biochemical and cellular role of Diphthong (Dpth). Work from G. Davis' lab implicates the polytopic membrane protein Dpth in synaptic homeostasis. The sequence of Dpth predicts a polytopic membrane with similarity to transporters, and we will now determine its subcellular location in neurons, and identify its substrates as described for Aim 2.